Head and neck cancer treatments that require the resection of glandular tissue in combination with irradiation therapy cause significant damage to target and surrounding tissues. Salivary glands are vulnerable and when compromised, a loss of function causes hyposalivation and 'dry mouth' (xerostomia) that lead to an increase in oral health risk and a decline in quality of life. Currently, there is no cure for xerostomia, only interventions for alleviating the discomfort associated with loss of salivary function. A novel therapy is underway to address this unmet need using hyaluronic acid (HA) hydrogels and primary cells from resected human salivary gland. Repeatable organization of salivary acini into functional secretory units is a key step toward the standardization of our model system. Our goal is to mimic the microenvironment of parotid salivary gland tissue in development to best support the organization of serous acini. Determining factors for organized acini are basement membrane (BM) deposition and lumen formation. Using live imaging light and fluorescence microscopy, we have observed the coordinated motility of acinar cells in HA hydrogels, prior to acini organization. In early stage organization, acinar cells are in a dynamic microenvironment continuously influenced by mechanical forces, and we hypothesize that mechanical forces drive the BM deposition, lumen formation, and structural integrity of the acini in 3D. In Aim 1, we intend to identify the signaling mechanisms involved in the net coordination of cell motility durin BM deposition and growth of the acini. Signaling mechanisms proposed in this coordination include integrin signaling at the cell-ECM interface, connexin mediated intra- and intercellular signaling, and nesprin4 nuclear repositioning. In Aim 2, we will measure the traction forces required to initiate the coordinated movement of a multicellular structure in 3D. Live-fluorescence imaging and computational modeling will be used to develop displacement field and cellular traction maps, and reconstruct cellular traction forces of organizing acini as a function of their size and microenvironment. In Aim 3, we will evaluate acini organization, lumen formation, and structural integrity in response to mechanical loading of the hydrogel. Effects of varying magnitude and frequency loads on acini organization and integrity will be evaluated. Successful completion of these specific aims will (1) standardize the model system used to engineer serous acini of the salivary parotid gland as well as the evaluation process for optimizing iterations of the HA hydrogel herein, (2) yield fundamental understanding of salivary acini structure/function relations, and (3) advance the translational potential of this tissue engineered system. Additionally, the PI, Dr. Danielle Wu, will gain experimental and computational training in 3D, crucial for her future as an independent researcher in tissue engineering, will acquire skills and perspective from operating at a multidisciplinary interface, and will advance her long-term career goals with training in manuscript and grant writing, mentorship, and collaboration skills.